Preparation of a macrocyclic lactone

ABSTRACT

Subject of the invention is a process for the preparation of a compound of formula (I) in which R 1 -R 9  represent, independently of each other hydrogen a substituent; m is 0, 1 or 2; n is 0, 1, 2 or 3; and the bonds marked with A, B, C, D, E and F indicate, independently of each other, that two adjacent carbon atoms are connected by a double bond, a single bond, a single bond and an epoxide bridge, or a single bond and a methylene bridge, including, where applicable, an E/Z isomer, a mixture of E/Z isomers, and/or a tautomer thereof, in each case in free form or in salt form, which process comprises I) bringing a compound of formula (II); wherein R 1 -R 7 , m, n, A, B, C, D, E and F have the same meanings as given for formula (I) above, into contact with a biocatalyst that is capable of specifically oxidising the alcohol at position 4″ in order to form a compound of formula (III), in which R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , m, n, A, B, C, D, E and F have the meanings given for formula (I); and 2) reacting the compound of formula (III) with an amine of the formula HN(R 8 )R 9 , wherein R 8  and R 9  have the same meanings as given for formula (I), and which is known, in the presence of a reducing agent.

The present invention relates to a process for the preparation of amacrocyclic lactone, a process for the preparation of the intermediatecompounds and the intermediate compounds used in the said process. Moreparticularly the invention relates to a process for the preparation acompound of the formula

in which

R₁-R₉ represent, independently of each other hydrogen or a substituent,

m is 0, 1 or 2;

n is 0, 1, 2 or 3; and

the bonds marked with A, B, C, D, E and F indicate, independently ofeach other, that two adjacent carbon atoms are connected by a doublebond, a single bond, a single bond and a epoxide bridge of the formula

or a single bond and a methylene bridge of the formula

including, where applicable, an E/Z isomer, a mixture of E/Z isomers,and/or a tautomer thereof, in each case in free form or in salt form,which process comprises,

1) bringing a compound of the formula

wherein

R₁-R₇, m, n, A, B, C, D, E and F have the same meanings as given forformula (I) above, into contact with a biocatalyst that is capable ofspecifically oxidising the alcohol at position 4″ in order to form acompound of the formula

in which R₁, R₂, R₃, R₄, R₅, R₆, R₇, m, n, A, B, C, D, E and F have themeanings given for formula (I); and

2) reacting the compound of the formula (II) with an amine of theformula HN(R₈)R₉, wherein R₈ and R₉ have the same meanings as given forformula (I), and which is known, in the presence of a reducing agent;

and, in each case, if desired, converting a compound of formula (I)obtainable in accordance with the process or by another method, or anE/Z isomer or tautomer thereof, in each case in free form or in saltform, into a different compound of formula (I) or an E/Z isomer ortautomer thereof, in each case in free form or in salt form, separatinga mixture of E/Z isomers obtainable in accordance with the process andisolating the desired isomer, and/or converting a free compound offormula (I) obtainable in accordance with the process or by anothermethod, or an E/Z isomer or tautomer thereof, into a salt or convertinga salt, obtainable in accordance with the process or by another method,of a compound of formula (I) or of an E/Z isomer or tautomer thereofinto the free compound of formula (I) or an E/Z isomer or tautomerthereof or into a different salt.

Methods of synthesis for the compounds of formula (I) are described inthe literature. It has been found, however, that the processes known inthe literature cause considerable problems during production basicallyon account of the low yields and the tedious procedures which have to beused. For example EP-A 0 465 121 discloses 4″-Keto- and4″-amino-4″-deoxy avermectin compounds and substituted amino derivativesthereof. The 4″ hydroxy group on the avermectin compounds are oxidizedto a ketone group or replaced with a (substituted) amino group. Thehydroxy groups at the 5 and 23-positions need to be protected in orderto avoid unwanted oxidations. The 4″-keto compound is aminated toprovide the compound corresponding to that of formula (I) above.Accordingly, the known processes are not satisfactory in that respect,giving rise to the need to make available improved preparation processesfor those compounds.

The compounds (I), (II) and (III) may be in the form of tautomers.Accordingly, herein-before and hereinafter, where appropriate thecompounds (I), (II) and (III) are to be understood to includecorresponding tautomers, even if the latter are not specificallymentioned in each case.

The compounds (I), (II) and (III) are capable of forming acid additionsalts. Those salts are formed, for example, with strong inorganic acids,such as mineral acids, for example perchloric acid, sulfuric acid,nitric acid, nitrous acid, a phosphoric acid or a hydrohalic acid, withstrong organic carboxylic acids, such as unsubstituted or substituted,for example halo-substituted, C₁-C₄alkanecarboxylic acids, for exampleacetic acid, saturated or unsaturated dicarboxylic acids, for exampleoxalic, malonic, succinic, maleic, fumaric or phthalic acid,hydroxycarboxylic acids, for example ascorbic, lactic, malic, tartaricor citric acid, or benzoic acid, or with organic sulfonic adds, such asunsubstituted or substituted, for example halo-substituted, C₁-C₄alkane-or aryl-sulfonic acids, for example methane- or p-toluene-sulfonic acid.Furthermore, compounds of formula (I), (II) and (III) having at leastone acidic group are capable of forming salts with bases. Suitable saltswith bases are, for example, metal salts, such as alkali metal oralkaline earth metal salts, for example sodium, potassium or magnesiumsalts, or salts with ammonia or an organic amine, such as morpholine,piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, forexample ethyl-, diethyl-, triethyl- or dimethyl-propyl-amine, or amono-, di- or tri-hydroxy-lower alkylamine, for example mono-, di- ortri-ethanolamine. In addition, corresponding internal salts may also beformed. Preference is given within the scope of the invention toagrochemically advantageous salts. In view of the close relationshipbetween the compounds of formula (I), (II) and (III) in free form and inthe form of their salts, any reference hereinbefore or hereinafter tothe free compounds of formula (I), (II) and (III) or to their respectivesalts is to be understood as including also the corresponding salts orthe free compounds of formula (I), (II) and (III), where appropriate andexpedient. The same applies in the case of tautomers of compounds offormula (I), (II) and (III) and the salts thereof. The free form isgenerally preferred in each case.

Preferred within the scope of this invention is a process for thepreparation of compounds of the formula (I), in which

n is 1;

m is 1;

A is a double bond;

B is single bond or a double bond,

C is a double bond,

D is a single bond,

E is a double bond,

F is a double bond; or a single bond and a epoxy bridge; or a singlebond and a methylene bridge;

R₁, R₂ and R₃ are H;

R₄ is methyl;

R₅ is C₁-C₁₀-alkyl, C₃-C₈-cycloalkyl or C₂-C₁₀-alkenyl;

R₆ is H;

R₇ is OH;

R₈ and R₉ are independently of each other H; C₁-C₁₀-alkyl orC₁-C₁₀-acyl; or together form —(CH₂)_(q)—; and

q is 4, 5 or 6.

Especially preferred within the scope of this invention is a process forthe preparation of a compound of the formula (I) in which

n is 1;

m is 1;

A, B, C, E and F are double bonds;

D is a single bond;

R₁, R₂, and R₃ are H;

R₄ is methyl;

R₅ is s-butyl or isopropyl;

R₆ is H;

R₇ is OH;

R₈ is methyl

R₉ is H.

Very especially preferred is a process for the preparation of Emamectin,more particularly the benzoate salt of Emamectin. Emamectin is a mixtureof 4″-deoxy-4″-N-methylamino avermectin B_(1a)/B_(1b) and is describedin U.S. Pat. No. 4,4874,749 and as MK-244 in Journal of OrganicChemistry, Vol. 59 (1994), 7704-7708. Salts of emamectin that areespecially valuable agrochemically are described in U.S. Pat. No.5,288,710. The compounds of the formula (I) are valuable pesticides,especially for combating insect and representatives of the orderAcarina. The pests mentioned include, for example, those that arementioned on page 5, lines 55 to 58, page 6 and page 7, lines 1 to 21 inEuropean Patent Application EP-A 736,252. The pests mentioned thereinare included thereto in the present subject matter of the invention.

The general terms used hereinbefore and hereinafter have the followingmeanings, unless defined otherwise:

Carbon-containing groups and compounds each contain from 1 up to andincluding 8, preferably from 1 up to and including 6, especially from 1up to and including 4, and more especially 1 or 2, carbon atoms.

Alkyl is either straight-chained, i.e. methyl, ethyl, propyl, butyl,pentyl or hexyl, or branched, e.g. isopropyl, isobutyl, sec-butyl,tert-butyl, isopentyl, neopentyl or isohexyl.

Alkeyl, both as a group per se and as a structural element of othergroups and compounds, for example haloalkenyl and arylalkenyl, is, ineach case taking due account of the number of carbon atoms contained inthe group or compound in question, either straight-chained, for examplevinyl, 1-methylvinyl, allyl, 1-butenyl or 2-hexenyl, or branched, forexample isopropenyl.

C₃-C₈Cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl,especially cyclohexyl.

Further aspects of the invention are

the compounds of the formula (III) as defined above; or

a process for the preparation of the compound of the formula (III)starting form the compound of the formula (II) according to step 1)above; or

a process for the preparation of the compound of the formula (I)starting form the compound of the formula (III) according to step 2)above.

Within the scope of the present invention, the term “biocatalyst” ismeant to include:

a) a living microorganism, for example in the form of vegetative cells,resting cells or freeze dried cells,

b) the spores of the said microorganism

c) a dead microorganism, preferably in a partially disintegrated form,that is to say with the cell wall/cell membrane mechanically orchemically or by spray drying permeabilized,

d) crude extracts of the cell contents of the said microorganism, and

e) an enzyme that converts the compounds of the formula (II) intocompounds of formula (III).

Bacteria and fungi are especially suitable microorganisms for theprocess according to the invention. Suitable bacteria are especiallyrepresentatives of Actinomycetes, especially of the genus Streptomyces.Preferred are strains of the genus Streptomyces selected from the groupconsisting of Streptomyces tubercidicus; Streptomyces chattanoogensis,Streptomyces lydicus, Streptomyces saraceticus and Streptomyceskasugaensis. The strains Streptomyces R-922 (Streptomyces tubercidicus),and especially Streptomyces I-1529 (Streptomyces tubercidicus), haveproven particularly suitable for the regiospecific oxidation of thehydroxy group at the 4″-position of compounds of the formula (II).

Streptomyces strains Streptomyces I-1529 and Streptomyces R-922, aredeposited pursuant to the provisions of the Budapest Treaty in theDSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1 b, D—38124 Braunschweig—Germany, under accessionnumber DSM-13135 and DSM-13136, respectively, on Nov. 5, 1999.

Additional strains have been identified which can be suitably used toperform the regiospecific oxidation according to the inventionincluding, for example, Streptomyces strain MAAG-7027 (Streptomycestubercidicus), Streptomyces strain DSM-40241 (Streptomyces saraceticus,also identified as Streptomyces chattanoogensis), Streptomyces strainNRRL-2433 (Streptomyces lydicus ssp. lydicus) and Streptomyces strainA/96-1208710 (Streptomyces kasugaensis). All the above strains areclosely related to Streptomyces strains Streptomyces I-1529 andStreptomyces R-922, respectively, which can be demonstrated by a 16srDNA analysis showing identities of between 99.4% and 100%.

The compounds of formula (I) are known to be highly active agents forcontrolling plant pests. In the known process for the preparation ofcompounds of the formula (I) as, for example, described in EP 301 806and also in the microbial process according to the invention, compoundsof formula (II) are used as starting materials.

In the known processes, in a first step the compounds of the formula(II) are protected on the oxygen in position 5, then oxidised to the4″-ketone, followed by conversion to the amine and deprotection of themasked hydroxy group in position 5, thereby employing conventionalprotecting group technology as described by Greene and Wuts, 1999(Protective Groups in Organic Synthesis).

The process according to the present invention has the advantage that itcomprises only two steps as compared with four of the known processes.It further avoids the use of protecting groups, and is ecologicallysafer since fewer chemicals have to be used. The compound of formula(III) resulting from the biocatalytic step according to the invention isknown, for example from EP 401 029.

In a specific embodiment the process according to the invention may, indetail, be carried out as follows:

In a first step compounds of the formula (III) are prepared. This can beaccomplished by directly contacting a compound of formula (II) with abiocatalyst that is capable of specifically oxidizing the alcohol atposition 4″ to a ketone of formula (III), and maintaining this contactfor a period of time that is sufficient for the oxidation reaction totake place.

Most expediently, the process is carried out by using a microorganism asthe biocatalyst, which microorganism is capable of carrying out theoxidation reaction according to the invention. Preferably, saidmicroorganism is cultured in a suitable cultivation medium promotingmicrobial proliferation and under controlled conditions in the presenceof a compound of the formula (II), and maintaining the joint incubationof the said microorganism and its substrate for a time sufficient forthe oxidation reaction to occur, preferably until from 25% to 99.9%,more preferably from 50% to 99.9% and most preferably from 80 to 99.9%of the added compound of the formula (II) has been converted intocompounds of the formula (III).

Alternatively and more preferably, the process is carried out by firstlyculturing a microorganism that is capable of carrying out the oxidationreaction according to the invention in a suitable cultivation mediumpromoting microbial proliferation and under controlled conditions, andthen harvesting the biomass of the microorganism by applying suitablemethods such as, for example, by filtration or centrifugation. Thebiomass of the microorganism is then either immediately used as abiocatalyst for the conversion of compounds of formula (II) intocompounds of formula (III) or may be stored in the cold either as suchor after freeze drying or spray drying before being used in thereaction. Said microorganism, either freshly harvested or stored asdescribed, and a compound of formula (II) are then jointly incubated ina reaction medium which does not favor microbial proliferation for atime sufficient for the oxidation reaction to occur, preferably untilfrom 25% to 99.9%, more preferably from 50% to 99.9% and most preferablyfrom 80 to 99.9% of the added compound of the formula (II), has beenconverted into compounds of the formula (III).

The reaction product of the formula (III) obtained in this manner may beseparated from starting material of the formula (II) without greattechnical expenditure by means of customary separating methods, forexample by fractional crystallisation or by chromatography.Chromatography includes, for example, column chromatography, thick layerchromatography or thin layer chromatography on mineral carrier materialssuch as silica gel or on organic exchanger resins.

Instead of vegetative cell structures, microbial spores may be usedwhich spores are harvested from microorganisms that are capable ofspecifically oxidizing the alcohol at position 4″ to a ketone of theformula (III), and are then incubated with a compound of the formula(II) for a period of time that is sufficient for the correspondingoxidation reaction to take place. The incubation of spores and substrateis preferably carried out in the absence of culture medium in order toprevent the spores from germinating.

Compounds of the formula (II) are used as a substrate for the oxidationreaction according to the invention. These compounds are known (see DE2717040) or can be prepared from known compounds analogously to knownprocesses. They are suitable for controlling pests in animals and plantsand are furthermore valuable starting materials or intermediates in thepreparation of compounds of the formula (I). The preparation ofcompounds of formula (III) can also be carried out by using for theoxidation of the compounds of formula (II) not the microorganism itselfbut active constituents originating from this microorganism (accordingto the definitions b) to a) above) that are capable of specificallyoxidizing the alcohol at position 4″ to a ketone of the formula (III).

Accordingly, a further aspect of the present invention is the use inimmobilised form of vegetative microorganism cells, cell-free extracts,spores, enzymes and mixtures of enzymes of the said microorganisms thatare capable of specifically oxidizing the alcohol at position 4″ to aketone of the formula (III).

The immobilisation of the said biocatalysts can be carried outanalogously to processes known per se. Within the scope of the presentinvention there may be mentioned especially processes that are based onadsorptive binding or ionic or covalent bonding of the said biocatalyststo solid, as a rule water-insoluble, carrier materials, on crosslinkingof biocatalysts by bi- or poly-functional reagents, on matrixencapsulation, on membrane separation or on a combination of two or moreof the above-mentioned processes.

The adsorptive binding to water-insoluble carriers (adsorbents) iscarried out especially by van der Waals forces. Numerous inorganic andorganic compounds and also synthetic polymers are suitable asadsorbents.

Methods for such an immobilisation of microorganisms are described byBickerstaff (Ed.), 1997 (Immobilisation of Enzymes and Cells), vanHaecht et al., 1985 (yeast cells/glass), Black et al., 1984 (yeastcells/refined steel, polyester), Wiegel and Dykstra, 1984(clostridia/cellulose, hemicellulose), Förberg and Häggström, 1984(clostridia/wood shavings) and also by Ehrhardt and Rehm, 1985(Pseudomonads/active carbon). Corresponding details for the use ofenzymes immobilised by adsorptive binding are to be found in Krakowiaket al., 1984 (glucoamylase/aluminium oxide), Cabral et al., 1984(glucoamylase/titanium-activated glass), Miyawaki and Wingard 1984(glucose oxidase/active carbon), Kato and Horikoshi, 1984 (glucosetransferase/synthetic resin) inter alia. Ionic bonds are based onelectrostatic attractions between oppositely charged groups of thecarrier material (such as, for example, commercially available ionexchangers, for example based on polysaccharides or on synthetic resins)and of the biocatalyst to be bound.

Methods of immobilising microorganisms based on ionic bonding aredescribed by DiLuccio and Kirwan, 1984 (Azotobacter spec./Cellex E(cellulose)) and by Giard et al., 1977 (animal cells/DEAE-Sephadex). Acorresponding immobilisation of enzymes can be carried out in accordancewith the details given by Angelino et al., 1985 (aldehydeoxidase/octylamino-Sepharose 4B), Hofstee, 1973 (lactatedehydrogenase/octylamino-Sephadex), Kühn et al., 1980 (glucoseoxidase/DEAE-Sephadex, DEAE-cellulose) and others.

A further method of immobilisation is based on the use of covalentbonding forces, which generally result in fixed linking of blocatalyststo one another or between biocatalyst and carrier material. Suitablecarrier materials are porous materials, such as glasses, silica or otherinsoluble inorganic materials.

Within the scope of the process according to the invention, themicroorganisms can be immobilised, for example, analogously to Messingand Oppermann, 1979 (Enterobacteria/borosilicate glass; yeastcells/zirconium oxide), Romanovskaya et al., 1981 (methanebacteria/Silochrome), Navarro and Durand, 1977 (yeast cells/poroussilica).

The immobilisation of enzymes can be carried out in accordance with themethod described by Weetall and Mason, 1973 (papain/porous glass) andMonsan et al., 1984 (invertase/porous silica).

In the process according to the invention not only are the carriermaterials already mentioned suitable for immobilisation but also a wholeseries of natural or synthetic polymers, such as, for example,cellulose, dextran, starch, agarose etc. or polymers, for example basedon acrylic and methacrylic acid derivatives, that are usually used inthe manufacture of reactive copolymers. Suitable reactive groups bymeans of which a bond to the biocatalyst is formed are reactivedinitrofluorophenyl or isothiocyanate groups, or especially oxirane andacid anhydride groups. A further possibility resides in the chlorideactivation of resins carrying carboxy groups, which are commerciallyavailable, for example, under the trade names Amberlite® XE-64 andAmberlite® IRC-50.

The immobilisation of microorganisms with the aid of natural orsynthetic carrier materials can be carried out as described by Chipley,1974 (Bacillus subtilis/agarose), Gainer et al., 1980 (Azotobacterspecies/cellulose), Jack and Zajic, 1977(Micrococcus/carboxymethylcellulose), Jirku et al., 1980 (yeastcells/hydroxyalkylmethacrylate) and also by Shimizu et al., 1975(bacterial cells/ethylene-maleic anhydride copolymer). Theimmobilisation of enzymes can be carried out analogously to Cannon etal., 1984 (lactate oxidase/cellulose), Dennis et al., 1984(chymotrypsin/Sepharose), Ibrahim et al., 1985 (epoxyhydrolase/dextran); Beddows et al., 1981 (α-galactosidase/nylon-acrylatecopolymer), Raghunath et al., 1984 (urease/methacrylate-acrylate), interalia.

In the crosslinking process, the biocatalysts are bonded to each otherby bi- or poly-functional reagents, such as glutardialdehyde,diisocyanates inter alia and form characteristically insoluble, usuallygelatinous aggregates of high molecular weight.

Such immobilisations of microorganisms can be carried out analogously toDe Rosa et al., 1981 (bacterial cells/co-crosslinking with eggalbumin bymeans of glutardialdehyde). Processes for the immobilisation of enzymesthat can be used within the scope of the present invention are describedby Barbaric et al., 1984 (invertase/crosslinking with adipic aciddihydrazide), Talsky and Gianitsopoulos, 1984 (chymotrypsin/peptide bondbetween the enzyme molecules without crosslinking agent), Workman andDay, 1984 (inulinase/crosslinking of the enzyme-containing cells withglutardialdehyde), Khan and Siddiqi, 1985 (pepsin/crosslinking withglutardialdehyde), Bachmann et al., 1981 (glucoseisomerase/co-crosslinking with gelatine by means of glutardialdehyde),Kaul et al., 1984 (α-galactosidase/co-crosslinking with egg albumin bymeans of glutardialdehyde).

Matrix encapsulation comprises the inclusion of the biocatalysts innatural or synthetic polymers, which are usually of gelatinousstructure. Matrix materials that are especially suitable for theinclusion of cells, organelles and spores are natural polymers such asalginate, carrageenan, pectin, agar, agarose or gelatine, since thesecompounds are non-toxic and protect the cells during handling. Alsosuitable are synthetic polymers, such as, for example, polyacrylamides,photo-crosslinked resins inter alia. The form of the matrixencapsulation is variable within wide limits and may include, forexample, spherical, cylindrical, fibrous and sheet forms. Theimmobilisation of microorganisms with the aid of natural or syntheticmatrix materials can be carried out as described by Mazumder et al.,1985 (bacterial cells/photo-crosslinked resins), Bettmann and Rehm, 1984(bacterial cells/polyacrylamide hydrazide), Umemura et al., 1984(bacterial cells/carrageenan), Karube et al., 1985 (bacterialprotoplasts/agar-acetylcellulose), Cantarella et al., 1984 (yeastcells/hydroxyethylmethacrylate), Qureshi and Tamhane, 1985 (yeastcells/alginate), Deo and Gaucher, 1984 (Hyphomycetes/carrageenan),Eikmeier and Rehm, 1984 (Hyphomycetes/alginate), Bihari et al., 1984(Hyphomycetes conidial/polyacrylamide), Vogel and Brodelius, 1984 (plantcells/alginate, agarose), Nakajima et al., 1985 (plant cells/agar,alginate, carrageenan).

The immobilisation of enzymes can be carried out analogously to Mori etal., 1972 (aminoacylase/polyacrylamide).

Membrane separation involves the creation of specific defined areas inwhich the reaction proceeds. The basic variants of membrane separationare differentiated as follows:

a) microencapsulation

b) liposome technique

c) the use of biocatalyst in membrane reactors.

The above-described immobilisation methods can be combined with oneanother, such as, for example, adsorption and crosslinking. In that casethe enzymes are first of all adsorbed on a carrier and then crosslinkedwith one another by a bifunctional reagent.

The incubation of the biocatalysts used within the scope of the presentinvention with compounds of the formula (II) for the specific oxidationof the alcohol at position 4″ to a ketone of the formula (III) can becarried out with the aid of processes such as those customary in appliedmicrobiology. In addition to the use of shake cultures there may bementioned especially various fermenter systems that have long beenestablished in microbiological research and industrial production.

The main task of the bioreactors is the creation of optimum hydrodynamicconditions in order to reduce the apparent Michaelis constants and toincrease the reaction speed.

This is essentially achieved by maintaining an adequate relativemovement between biocatalyst and surrounding medium, which increases theexternal mass transfer to such an extent that its hindrance in practiceno longer applies.

Types of reactors that are suitable for the process concerned include,for example, stirred vessel reactors, loop-type reactors, bed reactors,fluidised bed reactors, membrane reactors and also numerous specialforms of reactor, for example sieve-stirred reactors, rhomboid reactors,tube reactors inter alia (W. Hartmeier, Immobilisierte Biokatalysatoren,1986; W. Crueger and A. Crueger, Biotechnologie-Lehrbuch der angewandtenMikrobiologie, 1984; P. Präve et al., Handbuch der Biotechnologie,1984). The use of stirred vessel reactors is preferred within the scopeof the present invention.

Stirred vessel reactors are among the types of reactor most used in thebiotechnological art of fermentation. This type of reactor ensures arapid and thorough mixing of substrate and biocatalyst as a result ofhigh stirring capacities and a high oxygen transfer capacity.

The advantages of stirred vessel reactors reside in their simple andthus economical construction and in their well-researched properties.

In principle, when using stirred vessel reactors two kinds of operationare possible: first of all a “batch-type” operated process, theso-called “batch” process, and, secondly, a continuous process.

In the “batch” process the biocatalysts are removed by separation orfiltration once the process is complete and are either discarded(vegetative cells) or are used again in a second batch (immobilisedbiocatalysts).

When using the continuous process, there is a permanent continuousexchange of new substrate for the end product of the reaction. Thebiocatalysts must be prevented from leaving the reactor by means ofsuitable measures (sieve, filters, return devices).

The culturing of vegetative microorganism cells within the scope of thepresent invention is carried out according to known generally customarymethods, liquid nutrient media preferably being used for reasons ofpracticability.

The composition of the nutrient media varies depending on themicroorganism used. Generally, complex media with poorly defined,readily assimilable carbon(C) and nitrogen(N) sources are preferred, ascustomarily used, for example, also for the production of antibiotics.

In addition, vitamins and essential metal ions are necessary which,however, are as a rule contained in an adequate concentration asconstituents or impurities in the complex nutrient media used. Ifdesired, the said constituents such as, for example, essential vitaminsand also Na⁺, K⁺, Ca²⁺, Mg²⁺, NH₄ ⁺, (SO₄)2−, Cl⁻, (CO₃)²⁻ ions and thetrace elements cobalt and manganese and zinc, inter alia, may be addedin the form of their salts. Especially suitable nitrogen sources apartfrom yeast extracts, yeast hydrolysates, yeast autolysates and yeastcells are especially soya meal, maize meal, oat meal, edamine(enzymatically digested lactalbumin), peptone, casein hydrolysate, cornsteep liquors and meat extracts.

The preferred concentration of the said N-sources is from 0.1 to 6 g/l.Suitable carbon sources are, especially, glucose, lactose, sucrose,dextrose, maltose, starch, cerelose, cellulose, mannitol, malt extract,and molasses. The preferred concentration range of said carbon sourcesis from 1.0 to 25 g/l. The use of D-glucose, soluble starch or maltextract and also of cerelose as carbon source is of advantage for theoxidation process described in the following, especially if themicroorganisms used are representatives of the genus Streptomyces. Thus,for example, the following culture media are excellently suitable forrepresentatives of the genus Streptomyces:

Medium 1

1.0 g of soluble starch

0.2 g of peptone

0.2 g of yeast extract

adjust to 1 liter with distilled water, adjust to pH 7 with NaOH,autoclave.

Medium 2

4.0 g of D-glucose

10.0 g of malt extract

4.0 g of yeast extract

adjust to 1 liter with distilled water, adjust to pH 7 with NaOH,autoclave.

Medium 3

10.0 g of glycerol

20.0 g of dextrin

10.0 g of soytone (Difco Manual, 9th ed., Detroit, Difco Laboratories,1969)

2.0 g of (NH₄)₂SO₄

2.0 g of CaCO₃

adjust to 1 liter with distilled water, adjust to pH 7 with NaOH,autoclave.

Medium 4

10.0 g of D-glucose

10.0 g of malt extract

3.0 g of yeast extract

10.0 g of Pharmamedia (Traders Protein, Southern Cotton Oil Co., MemphisTenn., USA)

1.0 g of meat extract

adjust to 1 liter with distilled water, adjust to pH 7 with NaOH,autoclave.

Medium 5 (ISP-2 Agar)

yeast extract 4 g (Oxoid Ltd, Basingstoke, Hampshire, England)D(+)-glucose 4 g bacto malt extract 10 g (Difco No. 0186-17-7) agar 20 g(Difco No. 0140-01)

are dissolved in 1 l of demineralized water, and the pH is adjusted to7.0. The solution is sterilized at 121° C. for 20 min, cooled down andkept at 55° C. for the short time needed for the immediate preparationof the agar plates.

Medium 6 (PHG Medium)

peptone 10 g (Sigma 0521) yeast extract 10 g (Difco) D-(+)-glucose 10 gNaCl 2 g MgSO₄ × 7H₂O 0.15 g NaH₂PO₄ × H₂O 1.3 g K₂HPO₄ 4.4 g

are dissolved in 1 l of demineralized water, and the pH is adjusted to7.0.

The above-mentioned media are also excellently suitable for culturingrepresentatives of the genus Streptomyces and for carrying out theoxidation reaction. Both the above general data about the composition ofthe media, and also the media listed in detail herein, serve merely toillustrate the present invention and are not of a limiting nature.

Apart from the composition of the media, the procedure used to producethe media, such as, for example, the dissolving or suspending sequence,the sterilisation of the nutrient solution as a whole or thesterilisation of the individual constituents, the prevention ofcontamination inter alia, also plays a significant role and should beoptimised accordingly for the production process concerned.

It should also be noted that the sterilisation may cause alterations inthe pH value of the nutrient medium and also precipitations.

The remaining culturing methods also correspond to the processescustomarily used for culturing microorganisms.

On a small scale, the fermentations carried out within the scope of thepresent invention, including any precultures, are usually in the form ofshake cultures, in which case it is advantageous to use glass flasks offrom 0.1 to 5 liters, preferably from 0.5 to 5 liters capacity, whichcontain from 0.05 to 2 liters, preferably from 0.1 to 2 liters ofnutrient medium. The flasks are preferably equipped with a baffle. Afterautoclaving and adjusting the pH to values of from pH 4 to pH 8,especially from pH 7.0 to pH 7.5 (bacteria) or to values of from pH 6 topH 7.5 (fungi), the flasks are inoculated with the correspondingmicroorganism cultures under sterile conditions. The inoculationmaterial used is generally a preculture that has been produced frompreserved inoculation material in accordance with the data given below.

The cultures, including any precultures, are advantageously grown underaerobic conditions at a temperature of from about 25° C. to about 37°C., preferably about 26° C. to about 30° C., but especially at about 28°C., with continuous shaking at between about 80 rpm to about 300 rpm,preferably between about 100 rpm and 250 rpm, but especially at about120 rpm (revolutions per minute) on a rotatory shaking machine. Underthe above-mentioned conditions, with Streptomyces an optimum oxidationactivity has generally been reached after from 1.5 to 7 days' culturing.

Once the catalytic capacity of the cells is sufficiently high to carryout the desired oxidation reaction, preferably after 40 hours, thesubstrate (compounds of the formula (II)) is added, whereby themicroorganisms and the substance to be oxidized can be brought intocontact with one another in any manner. For practical reasons, it hasproved advantageous to add the substrate, that is to say a compound ofthe formula (II), to the microorganism in nutrient solution.

The substance to be oxidized can be used, for example, in powder form orin the form of a solution either in a suitable solvent such as, forexample, dimethylformamide, acetone, dimethyl sulfoxide,N-methyl-2-pyrrolidone or an alcoholic solvent such as, for example,methanol, ethanol, isopropanol or tert.-butanol, or an ether solventsuch as, for example, tetrahydrofuran or 1,4-dioxane (0.5 to 15% byvolume, preferably 2% by volume) or an ester solvent such as, forexample, ethyl acetate or a hydrocarbon solvent such as, for example,octane or cyclohexane or toluene or xylene, or in an a mixture of asuitable solvent and a suitable surfactant. The term “surfactant”comprises ionic, non-ionic and zwitterionic surfactants and will also beunderstood to include mixtures of surfactants.

Both water-soluble soaps and water-soluble synthetic surface-activecompounds are suitable anionic surfactants. Suitable soaps are thealkali metal salts, alkaline earth metal salts or unsubstituted orsubstituted ammonium salts of higher fatty adds (C₁₀-C₂₂), e.g. thesodium or potassium salts of oleic or stearic acid, or of natural fattyacid mixtures which can be obtained, e.g., from coconut oil or tallowoil. Further suitable surfactants are also the fatty acid methyltaurinsalts. More frequently, however, so-called synthetic surfactants areused, especially fatty sulfonates, fatty sulfates, sulfonatedbenz-imidazole derivatives or alkylarylsulfonates. The fatty sulfonatesor sulfates are usually in the form of alkali metal salts, alkalineearth metal salts or unsubstituted or substituted ammonium salts andcontain a C₁₀-C₂₂-alkyl radical which also includes the alkyl moiety ofacyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, ofdodecylsulfate, or of a mixture of fatty alcohol sulfates obtained fromnatural fatty acids. These compounds also comprise the salts of sulfatedand sulfonated fatty alcohol/ethylene oxide adducts. The sulfonatedbenzimidazole derivatives preferably contain 2 sulfonic acid groups andone fatty acid radical containing 8 to 22 carbon atoms. Examples ofalkylarylsulfonates are the sodium, calcium or triethanolamine salts ofdodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of acondensate of naphthalenesulfonic acid and form-aldehyde. Also suitableanionic surfactants are bile acid salts, e.g. the sodium salts of cholicacid or deoxycholic acid. Also suitable are corresponding phosphates,e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenolwith 4 to 14 moles of ethylene oxide; or phospholipids.

Suitable cationic surfactants are tetraalkyl ammonium salts, e.g. cetyltrimethylammonium bromide

Suitable neutral surfactants are alkyl glycosides, e.g alkyl-β-Dglucopyranosides, alkyl-β-D thioglucopyranosides, alkyl-β-D maltosidescontaining a C₆-C₁₂-alkyl radical. Further suitable neutral surfactantsare glucamides, e.g. N,N-bis(3-D-Gluconamidopropyl)-cholamide,N,N-bis(3-D-Gluconamidopropyl)-deoxycholamide, fatty acidN-methylglucamides containing a C₇-C₁₂-acyl radical. Further suitableneutral surfactants are mono- and polydisperse polyoxyethylenes, e.gBRIJ®, GENAPOL®, LUBROL®, PLURONIC®, THESIT®, TRITON®, TWEEN®.

Suitable zwitterionic surfactants are N,N,N-trialkyl glycines, e.g.N-n-dodecyl-N,N,-dimethylglycine. Further suitable zwitterionicsurfactants are ω-N,N,N-trialkylammonium alkyl sulfonates, e.g.3-(N-alkyl-N,N-dimethyl)-1-propane-sulfonate containing a C₈-C₁₆-alkylradical. Further suitable zwitterionic surfactants are3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate and3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propane-sulfonate.

The surfactants customarily employed in the art of solubilisation andformulation are described, inter alia, in the following publications:Bhairi S. M. (1997) “A guide to the Properties and Uses of Detergents inBiology and Biochemistry”, Calblochem-Novabiochem Corp., San DiegoCalif.; “1999 International McCutcheon's Emulsifiers and Detergents” TheManufacturing Confectioner Publishing Co., Glen Rock, N.J., U.S.A.

The course of the reaction is continuously monitored by chromatographicmethods generally used in microbiological research.

The present invention also relates to the culturing of microorganismsthat are capable of specifically oxidizing the alcohol at position 4″ toa ketone of the formula (III), and to the incubation thereof with thesaid compounds in bioreactors, especially in bioreactors of the stirredvessel reactor type. In order to ensure an optimum rate of productformation in the actual production fermenter, it is recommended that themicroorganisms first of all be multiplied in precultures. The number offermenter precultures depends on the inoculation material concentrationthat is optimum in each particular case. Advantageously, depending onthe microorganisms used, the following concentrations of inoculationmaterial are produced for a fermenter stage: bacteria 0.1-3%, fungi5-10%, Actinomycetales 5-10%.

The inoculation of small fermenters (up to 20 L) is usually carried outusing shaken flask precultures. In this case the total flask content isused to inoculate the fermenter. The starting material used for theproduction of precultures is usually preserved inoculation materialwhich may be, for example, in the form of lyophilisates, or of frozen orcold-stored material. The preserved inoculation material used within thescope of the present invention is preferably material stored at −80° C.

Multiplying the inoculation material is preferably carried out in liquidmedia in glass flasks on a rotatory shaking machine or, when usingspores, on solid nutrient substrates. The conditions relating tonutrient substrates and culturing parameters, such as temperature, pH,introduction of oxygen inter alia, must be optimised in accordance withthe microorganism or process used. The growth times for the preservedinoculation material vary from a few hours to several days depending onthe starting material used.

lyophilisates 3-10 days frozen preserved bacteria 4-18 hoursActinomycetales 1-5 days fungi 1-7 days cold-stored cultures bacteria4-24 hours Actinomycetales 1-3 days fungi 15 days

If spores are used as inoculation material, the spores are first of allmultiplied from preserved inoculation material on solid nutrientsubstrates under standardised conditions (sterile aeration, climaticchamber). If porous nutrient substrates based on peat, bran, rice orbarley are used, the cultures are shaken thoroughly daily to achievehigh spore densities. A further possibility lies in culturing thepreserved inoculation material on nutrient media solidified by agar orother customary gelling agents, it being preferable to use nutrientmedia that trigger the induction of spore formation.

The sporulation time is from 7 to 30 days depending on the microorganismused and on the nutrient medium used.

To inoculate the preculture- or production fermenters, the spores areeither suspended with surface-active agents, for example a Tween80(surfactant, available from Sigma-Aldrich Co., St. Louis, Mo. USA)solution, and transferred together with their nutrient medium into thefermenter or, if solid nutrient media are used, are washed off the solidnutrient substrates also using the said surface-active agents. Thespore-containing solution obtained in this manner is then used toinoculate the fermenters. Preferably, both the recovery of the sporesand the inoculation of the fermenters are carried out under sterileconditions.

To produce compounds of the formulae (III) within the scope of thepresent invention, bioreactors of various dimensions, embracingcapacities of the order of from 0.001 m³ to 450 m³, may be useddepending on the amount of product required.

If stirred vessel bioreactors are used, then the following fermentationparameters are to be considered as critical for the course of thereaction to be optimum:

1. Temperature: The biocatalytic oxidation reaction within the scope ofthe process according to the invention is preferably carried out in themesophilic temperature range (temperature range of from 20° C. to 45°C.). The optimum temperature range for growth and product formation isfrom 20° C. to 32° C., especially from 24° C. to 30° C.

2. Aeration: The aeration rate is from 0.1 to 2.5 vvm (volume of air pervolume of liquid per minute), preferably from 0.3 to 1.75 vvm. Theaeration rate must, if necessary, be adapted to the acquired oxygenrequirement in the course of the fermentation.

3. Pressure: Stirred vessel reactors are generally operated under slightexcess pressure of from 02 to 1.0 bar, preferably from 0.5 to 0.7 bar,in order to reduce the risk of contamination.

4. pH value: The pH value may vary within certain limits depending onthe microorganism used. If microorganisms from the Actinomycetes groupare used, the initial pH value is from pH 6 to pH 8, preferably from pH6.5 to pH 7.5.

If fungi are used, the initial pH of the culture solution is preferablyfrom pH 4 to pH 8, especially from pH 6 to pH 7.5.

5. Stirring: The stirring speed depends on the type of stirrer used andthe size of the fermenter. Within the scope of the present inventionstirrers with impellers of the disc type are preferred which, with astirred vessel reactor size of 0.002 m³, are operated at speeds of from150 rpm to 550 rpm, especially from 200 rpm to 500 rpm.

Within the scope of the present invention the duration of thefermentation varies from 20h to 10 days depending on the microorganismused. The biocatalytic reaction is discontinued when from about 25% toabout 99.9%, more preferably from about 50% to about 99.9% and mostpreferably from about 80% to about 99.9% of the substrate (compounds ofthe formula (II)) added at the beginning has been converted intocompounds of the formula (III).

To ascertain the optimum time for termination of the oxidation reaction,the course of the reaction is monitored for the whole of thefermentation by customary analysis processes, especially chromatographicprocesses, such as, for example, HPLC or thin layer chromatographicprocesses

In a modification of the above outlined process, the bioreactor may onlybe used for generating biomass, which is then harvested by filtration orcentrifugation. The biomass of the microorganism is then eitherimmediately used as a biocatalyst for the conversion of compounds offormula (II) into compounds of formula (III) or stored in the coldeither as such or after freeze drying or spray drying. Saidmicroorganism, either freshly harvested or stored as described, is thenfurther distributed to other vessels such as, for example, flaskspreferably equipped with baffles or to stirred vessel bioreactors,wherein the bioconversion process is carried out. The substrate(compounds of the formula (II)) is added, whereby the microorganism andthe substance to be oxidized can be brought into contact with oneanother in any manner. For practical reasons, a has proven advantageousto add the substrate, that is to say a compound of the formula (II), tothe microorganism in a buffered solution which does not favorproliferation. The substrate (compounds of the formula (II)) to beoxidized can be used, for example, in powder form or in the form of asolution in either a suitable solvent such as those described hereinpreviously.

In a preferred embodiment of the invention the substrate (compounds ofthe formula (II)) is first dissolved in a suitable solvent such as, forexample, dimethyl sulfoxide or Tween40 (surfactant, available fromSigma-Aldrich Co., St. Louis, Mo. USA) or a combination of both, andadded to baffled flasks containing a buffer solution, preferablyphosphate buffer, more preferably a phosphate buffer of 0.07M pH 7.0.The resulting solution is then sterilized before the biocatalyst(biomass of the microorganism) is added. This reaction mixture is thenincubated at room temperature and shaken at between 100 rpm and 150 rpm,preferably at about 120 rpm for about 2-7 days, depending on themicrobial strain.

In a further preferred embodiment of the invention the substrate(compounds of the formula (II)) is first dissolved in a suitable solventsuch as, for example, dimethyl sulfoxide or Tween40 (surfactant,available from Sigma-Aldrich Co., St. Louis, Mo. USA) or a combinationof both, and added to baffled flasks containing a cultivation mediumpromoting growth of the microorganism to be used for carrying out thedesired oxidation reaction. The resulting solution is then sterilizedbefore the biocatalyst (biomass of the microorganism) is added. Thisreaction mixture is then incubated at room temperature and shaken atbetween 100 rpm and 150 rpm, preferably at about 120 rpm for about 2-9days, depending on the microbial strain.

In a further specific embodiment of the invention a cell-free extract isprepared using wet cells, which are washed in a suitable buffersolution, resuspended in disruption buffer and disrupted by, forexample, mechanical means at a temperature of between 2° C. and 15° C.,preferably at a temperature of between 3° C. and 6° C. and mostpreferably at 4° C. The resulting suspension is centrifuged and thesupernatant cell free extract is collected.

To the so obtained cell free extract solutions are added comprising asuitable aliquot of a foreign electron supply system such as, forexample, ferredoxin and ferredoxin reductase and substrate. After theaddition of substrate the mixture is preferably immediately andthoroughly mixed and aerated. Then a suitable aliquot of NADPH is addedand the mixture incubated at a temperature of between 15° C. and 40° C.,preferably at a temperature of between 20° C. and 35° C. and mostpreferably at 30° C.

Processing of the fermentation broth in order to recover the oxidationproduct (compounds of the formula (III)) can be carried out by processescustomarily used in the art of fermentation (W. Crueger and A. Crueger,1984; P. Präve, 1984).

First of all, the particulate constituents are removed from the reactionbroth using filters, centrifuges or separators, to be extractedseparately from the filtrate.

If vegetative or dead cells are used as biocatalyst, and if a portion ofthe reaction products (compounds of the formula (III) are present insidethe cells, the cells must be disintegrated prior to extraction. For thispurpose various cell disintegration methods are available based onmechanical, thermal, chemical or enzymatic processes.

Mechanical methods suitable for use in the process according to theinvention are, for example, grinding in stirred ball mills or colloidmills, the use of pressure and relaxation in a homogenizer and celldisintegration by the action of ultrasound. Non-mechanical processesinclude cell disintegration by drying, lysis of the cells by osmoticshock, chemical autolysis and enzymatic lysis of the cells.

Once the particulate constituents have been removed, the reactionproducts are concentrated by extracting the culture solution and theseparated cellular constituents with suitable solvents. For theextraction there are also numerous aids available that are customarilyused in the art of fermentation, such as, for example, mixer-settlers,countercurrentcolumns, extraction centrifuges, among others.

It is also possible to concentrate the reaction products, for example,by membrane filtration, ultrafiltration, freeze concentration, ionexchange processes, among others.

The further processing of the crude product obtained after theextraction can be carried out by methods that are well established inmicrobiological and chemical research and in industrial use.

These processes include, for example, chromatographic methods,such asadsorption chromatography, ion exchange chromatography,molecular sievechromatography, affinity chromatography, hydrophobic chromatography,partition chromatography, covalent chromatography and others, but inaddition to these also various crystallisation processes.

Solvents suitable for extraction, either as such or as mixtures thereofare: aromatic hydrocarbons such as toluene, xylene mixtures orsubstituted naphthalenes, phthalates such as dibutyl phthalate ordioctyl phthalate, aliphatic hydrocarbons such as isomers of hexane,heptane, octane or paraffins or cyclohexane, alcohols and glycols andtheir ethers and esters, such as methanol, ethanol, 2-propanol,1-butanol, tert.butanol, ethylene glycol, methyl tert.butyl ether, ethylacetate, ethylene glycol monomethyl or monoethyl ether, ketones such asacetone or 2-butanone or cyclohexanone, chlorinated hydrocarbons such asdichloromethane or chloroform or carbon tetrachloride.

The term “surfactants” will also be understood to include mixtures ofsurfactants. Both water-soluble soaps and water-soluble syntheticsurface-active compounds are suitable anionic surfactants. Suitablesoaps are the alkali metal salts, alkaline earth metal salts orunsubstituted or substituted ammonium salts of higher fatty acids(C_(10-C) ₂₂), e.g. the sodium or potassium salts of oleic or stearicacid, or of natural fatty acid mixtures which can be obtained, e.g.,from coconut oil or tallow oil. Further suitable surfactants are alsothe fatty acid methyltaurin salts. More frequently, however, so-calledsynthetic surfactants are used, especially fatty sulphonates, fattysulphates, sulphonated benz-imidazole derivatives oralkylarylsulphonates. The fatty sulphonates or sulphates are usually inthe form of alkali metal salts, alkaline earth metal salts orunsubstituted or substituted ammonium salts and contain a C₁₀-C₂₂-alkylradical which also includes the alkyl moiety of acyl radicals, e.g. thesodium or calcium salt of lignosulphonic acid, of dodecylsulphate, or ofa mixture of fatty alcohol sulphates obtained from natural fatty acids.These compounds also comprise the salts of sulphated and sulphonatedfatty alcohol/ethylene oxide adducts. The sulphonated benzimidazolederivatives preferably contain 2 sulphonic acid groups and one fattyacid radical containing 8 to 22 carbon atoms. Examples ofalkylarylsulphonates are the sodium, calcium or triethanolamine salts ofdodecylbenzenesulphonic acid, dibutylnaphthalenesulphonic acid, or of acondensate of naphthalenesulphonic acid and form-aldehyde. Also suitableare corresponding phosphates, e.g. salts of the phosphoric acid ester ofan adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide; orphospholipids. The surfactants customarily employed in the art offormulation are described, inter alia, in the following publication:“1986 International McCutcheon's Emulsifiers and Detergents” TheManufacturing Confectioner Publishing Co., Glen Rock, N.J., U.S.A.

A preferred embodiment of the invention is a method to produce4″-oxo-avermectin by bringing a biocatalyst such as a microorganismcapable of converting avermectin to 4″-oxo-avermectin into contact withavermectin and isolating the produced 4″-oxo-avermectin from thereaction mixture.

An embodiment of the invention is a method to produce a compound offormula (III), which preferably is 4″-oxo-avermectin, which comprisesthe following steps

(1) production of cells by inoculation of a nutrient media promotingcell growth with precultures of a microorganism capable of converting acompound of formula (II) to a compound of formula (III), preferablyavermectin to 4″-oxo-avermectin;

(2) harvesting of the cells after growth

(3) dissolving a compound of formula (II), preferably avermectin, in anappropriate solvent

(4) addition of the solution from step (3) to a reaction medium whichdoes not promote cell proliferation

(5) addition of cells from step (2) to the reaction medium from step (4)

(6) shaking or stirring of the reaction mixture of step (5) in thepresence of air

(7) separation of the cells from the medium

(8) extraction of the supernatant and of the cells with appropriatesolvents

(9) concentration of the organic solvent phases from step (8)

(10) purification of a compound of formula (III), which preferably is4″-oxo-avermectin, contained in the extract (9) by chromatography orcrystallisation

A further preferred embodiment of the invention is a method to produce acompound of formula (III), which preferably is 4″-oxo-avermectin, whichcomprises the following steps:

(1) production of cells by inoculation of a nutrient media promotingcell growth with precultures of a microorganism capable of converting acompound of formula (II) to a compound of formula (III), preferablyavermectin to 4″-oxo-avermectin;

(2) harvesting of the cells after growth

(3) dissolving a compound of formula (II), preferably avermectin, in anappropriate solvent

(4) addition of the solution from step (3) to a reaction medium whichdoes not promote cell proliferation

(5) addition of cells from step (2) to the reaction medium from step (4)

(6) shaking or stirring of the reaction mixture of step (5) in thepresence of air

(7) whole broth extraction with an appropriate solvent

(8) phase separation

(9) concentration of the solvent phase from step (8) in vacuo

(10) purification of a compound of formula (III), which preferably is4″-oxo-avermectin, contained in the extract (9) by chromatography orcrystallisation

A further preferred embodiment of the invention is a method to produce acompound of formula (III), which preferably is 4″-oxo-avermectin whichcomprises the following steps:

(1) dissolving a compound of formula (II), which preferably isavermectin, in an appropriate solvent

(2) addition of the solution from step (1) to a nutrient media promotingcell growth

(3) inoculation of the nutrient media of step (2) with precultures of amicroorganism capable of converting a compound of formula (II) to acompound of formula (III), preferably avermectin to 4″-oxo-avermectin;

(4) cultivation of a microorganism capable of converting a compound offormula (II) to a compound of formula (III), preferably avermectin to4″-oxo-avermectin;

(5) separation of the cells from the medium

(6) extraction of the supernatant and of the cells with appropriatesolvents

(7) concentration of the organic solvent phases from step (6) in vacuo

(8) purification of a compound of formula (III), which preferably is4″-oxo-avermectin, contained in the extract (7) by chromatography orcrystallisation.

A further preferred embodiment of the invention is a method to produce acompound of formula (III), which preferably is 4″-oxo-avermectin, whichcomprises the following steps:

(1) dissolving a compound of formula (II), preferably avermectin, in anappropriate solvent

(2) addition of the solution from step (1) to a nutrient media promotingcell growth

(3) inoculation of the nutrient media of step (2) with precultures of amicroorganism capable of converting avermectin to 4″-oxo-avermectin

(4) cultivation of a microorganism capable of converting a compound offormula (II) to a compound of formula (III), preferably avermectin to4″-oxo-avermectin;

(5) whole broth extraction with an appropriate solvent

(6) phase separation

(7) concentration of the solvent phase from step (6) in vacuo

(8) purification of a compound of formula (III), which preferably is4″-oxo-avermectin, contained in the extract (7) by chromatography orcrystallisation

In a second purely chemical step the so obtained compound of the formula(III) can be reacted with an amine of the formula HN(R₈)R₉, wherein R₈and R₉ have the same meanings as given for formula (I), and which isknown, in the presence of a reducing agent.

The reaction components can be reacted in the absence of a solvent,preferably however in the presence of a solvent. Another possibilityconsists in carrying out the reaction in an excess of one of thereaction partners, especially in a liquid amine. Usually the addition ofan inert liquid solvent or diluent is however advantageous. Examples ofsuch solvents or diluents are aromatic, aliphatic and alicyclichydrocarbons and halogenated hydrocarbons, such as benzene, toluene,xylene, mesitylene, tetraline, chlorobenzene, dichlorbenzene,brombenzene, petrolether, hexane, cyclohexane, dichlormethane,trichlormethane, tetrachlormethane, dichlorethane, trichlorethene ortetrachlorethene; esters, such as acetic acid ethylester; ethers, suchas diethylether, dipropylether, diisopropylether, dibutylether,tert.-butylmethylether, ethylenglycolemonomethylether,ethylenglycolemonoethylether, ethylenglycoledimethylether,dimethoxydiethylether, tetrahydrofurane or dioxane; ketones, such asacetone, methylethylketone or methylisobutylketon; alcohols, such asmethanol, ethanol, propanol, isopropanol, butanol, ethylenglycol orglycerine; amides, such as N,N-dimethylformamide, N,N-diethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone or hexamethyl phosphoric acidtriamide; nitriles, such as acetonitrile or propionitrile; andsulfoxides, such as dimethylsulfoxide; organic acids, such as aceticacid; and water.

Preferred solvents are ethers such as tetrahydrofurane andethylenglycoledimethylether, especially tetrahydrofurane; alcoholes suchas methanol, ethanol or isopropanol; halogenated solvents such asdichloromethane or dichlorethane; aromatic solvents such as benzene ortoluene; nitriles such as acetonitrile, amides such asN,N-dimethylformamide, carbonic acids such as acetic acid; water; andmixtures thereof.

Very especially preferred solvents are methanol or ethanol or mixturesthereof.

The reaction is preferrably carried out in a pH-range of between 0 and14, especially between 2 and 10, in many cases between 6 and 9, veryespecially at pH 9.

The reaction is preferrably carried out in a temperature range ofbetween −80° C. and +140° C., preferred between −30° C. and +100° C., inmany cases between −10° C. and +80° C., especially between 0° C. and+50° C.

Preferred reducing agents are hydrides such as borohydrides; boranes;formic acid; formiates; or hydrogen. Especially preferred are hydridessuch as sodiumborohydride, zincborohydride, lithiumborohydride,sodiumcyanoborohydride, sodiumtriacetoxyborohydride ortetramethyl-ammoniumtriacetoxyborohydride. Especially preferred issodiumborohydride.

The reaction can be carried out—where applicable—in the presence ofcertain further chemicals such as a homogeneous or heterogeneouscatalysts or acids. Especially suitable are acids such as hydrochloricacid, p-toluenesulfonic acid, acetic acid, propionic acid, tartaric acidor phthalic acid; Lewis acids such as for example titaniumtetrachloride,titaniumtetraisopropylate or zinc chloride; salts such as for examplemagnesiumperchlorate, sodiumacetate, sodium-potassiumtartrate,ytterbiumchloride or pyridinium-p-toluene-sulfonate; water absorbingagents such as for example sodiumsulfate, molecular sieve or silicagel;or mixtures thereof. Preferred additional agents are acids such asacetic acid, propionic add or tartaric acid; preferred is acetic acid.When the reduction is carried out with hydrogen, the addition of one orseveral suitable homogeneous or heterogeneous catalysts is advantageous.Preferred such catalysts are heterogeneous metal catalysts which areknown in the art, preferrably Ni-, Pt- or Pd-catalysts, especiallyRaney-nickel and Lindlar catalyst (Pd—CaCO₃—PbO). Suitable homogeneouscatalysts are especially Rhodium complexes such as Wilkinsons catalysts(Chloro-tris-triphenyl-rhodium).

The compounds of formula (I), in each case in free form or in salt form,may be in the form of one of the possible isomers or in the form of amixture thereof, for example depending on the number of asymmetriccarbon atoms in the molecule and the absolute and relative configurationthereof and/or depending on the configuration of non-aromatic doublebonds in the molecule they may be in the form of pure isomers, such asantipodes and/or diastereoisomers, or in the form of mixtures ofisomers, such as mixtures of enantiomers, for example racemates,mixtures of diastereoisomers or mixtures of racemates; the inventionrelates both to the pure isomers and to all possible mixtures of isomersand this is to be understood hereinbefore and hereinafter, even ifstereochemical details are not specifically mentioned in each case.

Mixtures of diastereoisomers and mixtures of racemates of compounds offormula (I), or salts thereof, obtainable in accordance with the processdepending on the starting materials and procedures chosen or by othermeans, can be separated on the basis of the physicochemical differencesbetween the constituents into the pure diastereoisomers or racemates inknown manner, for example by fractional crystallisation, distillationand/or chromatography.

Mixtures of enantiomers, such as racemates, so obtainable can beresolved into the optical antipodes by known methods, for example byrecrystallisation from an optically active solvent, by chromatography onchiral adsorbents, e.g. high-pressure liquid chromatography (HPLC) onacetyl cellulose, with the aid of suitable microorganisms, by cleavagewith specific immobilised enzymes, via the formation of inclusioncompounds, e.g. using chiral crown ethers, in which case only oneenantiomer is complexed, or by conversion into diastereoisomeric salts,e.g. by reaction of a basic end product racemate with an opticallyactive acid, such as a carboxylic acid, e.g. camphoric, tartaric ormalic acid, or a sulfonic acid, e.g. camphorsulfonic acid, andseparation of the resulting mixture of diastereoisomers, e.g. on thebasis of their different solubilities by fractional crystallisation,into the diastereoisomers from which the desired enantiomer can be freedby the action of suitable, e.g. basic, agents.

Apart from by separation of corresponding mixtures of isomers, it ispossible according to the invention to obtain pure diastereoisomers orenantiomers also by generally known methods of diastereoselective orenantioselective synthesis, for example by carrying out the processaccording to the invention using starting materials having acorrespondingly suitable stereochemistry.

The compounds of formulae (I) and (III), acid addition products and thesalts thereof can also be obtained in the form of their hydrates and/orcan include other solvents, for example solvents which may have beenused for the crystallisation of compounds occurring in solid form.

The invention relates to all those forms of the process according towhich a compound obtainable as starting material or intermediate at anystage of the process is used as starting material and all or some of theremaining steps are carried out, or a starting material is used in theform of a derivative or a salt and/or its racemates or antipodes or,especially, is formed under the reaction conditions.

Compounds of formula (I) and (III) obtainable in accordance with theprocess or by other means may be converted into different compounds offormula (I) and (III) in a manner known per se.

In the process of the present invention there are preferably used thosestarting materials and intermediates, in each case in free form or insalt form, which result in the compounds of formulae (I) and (III), orsalts thereof, described at the beginning as being especially valuable.

In the case R₉ is hydrogen, the reaction step 2) may be split into twoseparate steps, wherein in a first step, a compound of the formula

in which R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, m, n, A, B, C, D, E and F havethe meanings given for formula (I) above, is formed by reaction of acompound of the formula (III) with a compound of the formula H₂N(R₈),wherein R₈ has the same meanings as given for formula (I) above, and ina second step, the said compound of the formula (IV) is reducedaccording to the procedure of step 2) above. The said two individualsteps may be carried out in a one pot synthesis without isolating thecompound of the formula (IV); It may however be anevatageous to isolatethe compound (IV), for instance for purification purposes. The compoundsof the formula (IV) are novel and are also an aspect of the presentinvention.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. The invention relates especially to the preparation processesdescribed in the Examples.

Example 1 Cell Production

1.1 Streptomyces tubercidicus Strain I-1529

Precultures of strain I-1529 (Streptomyces tubercidicus; DSM-13135) aregrown in 20 500 ml baffled Erlenmeyer flasks, each containing 100 ml ofmedium 2, with orbital shaking at 120 rpm at 28° C. for 3 days.

These cultures are used to inoculate a 50 liter fermenter containing 401 medium 4. The cells were grown at 28° C. with an aeration of 0.7 vvm(=30 liter/min). The stirrer speed was maintained between 200 rpm and300 rpm, guided by a pO₂-sensor, to prevent pO₂ falling below 25%. After2 days of growth, the cells are harvested by centrifugation, using aflow-through centrifuge. 4.2 kg cells (wet) are obtained.

1.2 Streptomyces tubercidicus Strain R-922

Streptomyces tubercidicus Strain R-922 (DSM-13136) is grown in a Petridish on ISP-2 agar (medium 5). This culture is used to inoculate 4 500ml shake flasks with baffle, each containing 100 ml PHG medium (medium6). These pre-cultures are grown on an orbital shaker with 120 rpm at28° C. for 96 h and then used to inoculate a 10 liter fermenter equippedwith a mechanical stirrer and containing 8 liter PHG medium. This mainculture is grown at 28° C. with stirring at 500 rpm and an aeration of1.75 vvm (14 l/min) and a pressure of 0.7 bar. At the end of theexponential growth, after about 20 h, the cells are harvested bycentrifugation. The yield of wet cells is 70-80 g/l culture. For furtherprocessing, the wet cells can be stored at 4° C., preferably not longerthan a week.

Example 2 Reaction Procedure

2.1 Resting Culture

2.1.1 Reaction Conditions

35.5 g avermectin (techn.) are dissolved in 1.05 l dimethylsulfoxide/Tween40 1:1. This solution is distributed by adding aliquotsof 25 ml to 42 3 l-Erienmeyer flasks with baffle, each containing 1 l ofreaction medium. These solutions are sterilized at 121° C. for 20 min.After cooling to room temperature, 100 g wet cells (fresh, or stored at4° C. for not longer than 4 days), as prepared in Example 1.1 and 1.2,respectively, are added. Subsequently these reaction mixtures are shakenat room temperature with 120 rpm for 4-5 days.

Reaction Medium:

0.5 g molasses

0.5 g MgCl₂

12.5 mg ZnCl₂

12.5 mg MnCl₂.4H₂O

25 mg CoCl₂.6H₂O

12.5 mg NiCl₂.6H2O

2.5 mg CuCl₂.2H2O

6.3 mg NaMoO₄.2H₂O

0.15 ml 1M HCl

adjust to 1 liter with phosphate buffer 70 mM pH 6.0, autoclave.

2.1.2 Work Up

The reaction mixtures are centrifuged in 500 ml polypropylene centrifugeflasks at 4° C. for 15 min at 13000×g.

The supernatants from the 40 l reaction mixture are pooled and extractedtwice with methyl tert.butyl ether (0.5 vol. eq., 0.4 vol. eq.). Thepooled methyl tert.butyl ether phases are then back-extracted threetimes with 0.185 vol. eq. distilled water. The methyl tert.butyl etherphase is concentrated in vacuo on the rotary evaporator. Drying of theresidue yields 10-12 g extract S. The aqueous phases are discarded.

The centrifuged cells from 120 to 132 centrifuge flasks are extracted asfollows:

Cells from 24 centrifuge flasks are transferred to a 2 l-Erienmeyerflask. To each Erienmeyer flask are added 80 g of diatomeous earth(Hyflo Supercell®, purified) and 1.2 l acetone. After manual mixing, themixture is homogenized by means of a large magnetic stirbar. Theresulting pulp is vacuum filtered through paper on a 20 cmØ Büchnerfunnel and washed with acetone until colorless elution. Thus filtrate C1and filter cake C1 are obtained.

Filtrate C1 is concentrated in vacuo on a rotary evaporator to removethe acetone. The resulting aqueous phase is then extracted three timeswith 0.7 l toluene. The combined toluene phases are dried over anhydroussodium sulfate. Filtration and evaporation on the rotary evaporator invacuo yields extract C1.

Filter cake C1 is transferred to a 2 l-Erienmeyer flask and manuallymixed with 1.5 L toluene. The mixture is homogenized by means of a largemagnetic stirbar. The resulting pulp is vacuum filtered through paper ona 20 cmØ Büchner funnel and washed with toluene until colorless elution.Thus filtrate C2 and filter cake C2 are obtained. Filter cake C2 isdiscarded.

Filtrate C2 is concentrated in vacuo on a rotary evaporator to yieldextract C2 which is dried in high vacuum.

The combined extracts C1 and C2 from the 40 l reaction mixture are driedin high vacuum to yield 30-35 g extract C.

45 g of combined exacts S & C are flash chromatographed analogously tothe description of Clark-Still et al. on a column packed with 1.5 kgsilica gel (Merck 60, 0.040-0.063 mm) by elution with ethylacetate:hexane 3:2 at 0.5 bar N₂-pressure and monitoring with thin layerchromatography. The yield of pure 4″-oxo-avermectin is 5.6 g.

2.1 Proliferating Culture

2.2.1 Reaction Conditions

1 g avermectin (techn.) is dissolved in 50 ml dimethyl sulfoxide/Tween401:1. This solution is distributed by adding aliquots of 2.5 ml to 20 500ml-Erlenmeyer flasks with baffle, each containing 100 ml of medium 4.These solutions are sterilized at 121° C. for 20 min. After cooling toroom temperature, 5 ml of preculture, as prepared in Example 1.1 and1.2, respectively, are added. Subsequently these inoculated cultureswere incubated at 28° C. for 7 days with orbital shaking at 120 RPM.

2.2.2 Work Up

The reaction mixtures are centrifuged in 500 ml polypropylene centrifugeflasks at 4° C. for 15 min at 13000×g and analogously processed asdescribed in Example 3. 252 mg pure 4″-oxo-avermectin are obtained.

2.3 Cell-free Biocatalysis

2.3.1 Preparation of Cell Free Extract

Stock Solutions:

PP-buffer: 50 mM K₂HPO₄/KH₂PO₄ (pH 7.0)

Disruption buffer: 50 mM K₂HPO₄/KH₂PO₄ (pH 7.0).

5 mM benzamidine,

2 mM dithiothreitol,

0.5 mM Pefabloc (from Roche Diagnostics)

Substrate: 10 mg avermectin are dissolved in 1 ml isopropanol.

6 g wet cells, washed in PP-buffer are resuspended in 35 ml disruptionbuffer and disrupted in a French press at 4° C. The resulting suspensionis centrifuged for 1 h at 35000×g. The supernatant cell free extract iscollected.

2.3.2 Development of an Assay for Enzyme Activity

Stock Solutions:

Ferredoxin solution 5 mg ferredoxin (from spinach) 1-3 mg/ml inTris/HCl-buffer (from Fluka)

or solution 5 mg ferredoxin (from Clostridium pasteurianum) 1-3 mg/ml inTris/HCl-buffer (from Fluka)

or solution 5 mg ferredoxin (from Porphyra umbillicalis) 1-3 mg/ml inTris/HCl-buffer (from Fluka)

Ferredoxin Reductase: solution of 1 mg freeze dried ferredoxin reductase(from spinach) 3.9 U/mg in 1 ml H₂O (from Sigma)

NADPH: 100 mM NADPH in H₂O (from Roche Diagnostics)

(all stock solutions were stored at −20° C. and when in use they werekept on ice)

HPLC Conditions:

HPLC instrument: Merck-Hitachi

HPLC-column: 70×4 mm, Kromasil 100 C18, 3.5μ (from Macherey-Nagel,Switzerland)

solvent A: acetonitrile, containing 0.0075% trifluoroacetic acid

solvent B: water, containing 0.01% trifluoroacetic acid

flow: 1.5 ml/min

detection: UV 243 nm

sample: 30 μl

Retention times: avermectin B1a 3.18 min

4Δ-oxo-avermectin B1a 4.74 min

Pump table: 0.0 min  75% A 25% B linear gradient to 7.0 min 100% A  0% B9.0 min 100% A  0% B step to 9.1 min  75% A 25% B 12.0 min  75% A 25% B

To 475 μl cell free extract the following solutions are added 10 μlferredoxin, 10 μl ferredoxin reductase and 1 μl substrate. After theaddition of substrate the mixture is immediately and thoroughly mixedand aerated. Then 5 μl of NADPH are added and the mixture incubated at30° C. for 30 min. Then, 1 ml methyl-t-butyl ether is added to thereaction mixture and thoroughly mixed. The mixture is centrifuged for 2min at 14000 rpm, and the methyl-t-butyl ether phase is transferred intoa 10 ml flask and evaporated in vacuo by means of a rotary evaporator.The residue is dissolved in 200 μl acetonitrile and transferred into anHPLC-sample vial. Upon injection of a 30 μl sample a peak appeared at4.74 min, indicating the presence of 4″-oxo-avermectin B1a. A mass of870 Da can be assigned to this peak by HPLC-mass spectrometry whichcorresponds to the molecular weight of 4″-oxo-avermectin B1a.

When analyzing product formation by HPLC and HPLC-mass spectrometry, asecond peak appears at 2.01 min corresponding to ketohydrate4″-hydroxy-avermectin. This is an indication that the cell-free extractconverts avermectin by hydroxylation to 4″-hydroxy-avermectin from which4″-oxo-avermectin is formed by dehydration.

The spinach ferredoxin can be replaced by, for example, ferredoxin fromthe bacterium Clostridium pasteurianum or from the red alga Porphyraumbilicalis, which also result in the biocatalytic conversion ofavermectin to 4″-oxo-avermectin.

Example 3 Steptomyces Strains

Strains of the genus Streptomyces that can be used in the processaccording to the invention and their relationship to S tubercidicusstrains I-1529 and R-922 based on a 16s rDNA analysis can be seen fromthe following table.:

% 16s Strain Closest GenBank rDNA Number Match Identity I-1629Streptomyces tubercidicus DSM 40261 Type 100 strain R-922 Streptomycestubercidicus DSM 40261 Type 100 strain MAAG-7027 Streptomycestubercidicus DSM 40261 Type 100 strain DSM-40241 (listed as Streptomycessaraceticus = 100 ATCC-25496) Streptomyces chattanoogensis 99.8Streptomyces lydicus ATCC 25470 = NRRL-2433 A/96-1208710 Streptomyceskasugaensis DSM 40819 Type 99.5 strain 99.4 Streptomyces kasugaensisM338-M1 NRRL-2433 (listed as Type strain Streptomyces lydicus ssplydicus = ATCC 25470 = CBS 703.69 = DSM 40461)

Example 4 Preparation of 4″-Deoxy-4″-epi-(methylamino)-avermectin B1 ofthe Formula

wherein R is methyl and ethyl;

3 ml acetic acid in 30 ml methanol are cooled to 0 to 5° C. Gaseousmethylamine is added until the pH of the solution is 9.

To 8.25 ml of this solution of methylamine is added at 0° C. a solutionof 1.0 g 4″-Oxo-avermectin B1 in 6 ml methanol. The mixture is allowedto warm up to ambient temperature and then stirred for a further 50minutes at room temperature. Then, 90 mg sodiumborohydride in 2.5 mlethanol are added and the resulting mixture stirred for another 45minutes. 10 ml ethylacetate are added to the reaction mixture, theorganic phase extracted three times with saturated aqueoussodiumhydrogencarbonate solution, the organic phase separated and driedover sodiumsulfate. The solvent is distilled off yielding4″-Deoxy-4″-epi-(methylamino)-avermectin B1. The purity is over 90%.

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What is claimed is:
 1. A process for the preparation of a compound ofthe formula

in which R₁-R₉, represent, independently of each other hydrogen or asubstituent; m is 0, 1 or 2; n is 0, 1, 2 or 3; and the bonds markedwith A, B, C, D, E and F indicate, independently of each other, that twoadjacent carbon atoms are connected by a double bond, a single bond, asingle bond and a epoxide bridge of the formula

or a single bond and a methylene bridge of the formula

including, where applicable, an E/Z isomer, a mixture of E/Z isomers,and/or a tautomer thereof, in each case in free form or in salt form,which process comprises 1) bringing a compound of the formula

wherein R₁-R₇, m, n, A, B, C, D, E, and F have the same meanings asgiven for formula (I) above, into contact with a biocatalyst that iscapable of specifically oxidising the alcohol at position 4″ in order toform a compound of the formula

in which R₁, R₂, R₃, R₄, R₅, R₆, R₇, m, n, A, B, C, D, E and F have themeanings given for formula (I); and 2) reacting the compound of theformula (III) with an amine of the formula HN(R₈)R₉, wherein R₈ and R₉have the same meanings as given for formula (I), and which is known, inthe presence of a reducing agent; and, in each case, if desired,converting a compound of formula (I) obtainable in accordance with theprocess or by another method, or an E/Z isomer or tautomer thereof, ineach case in free form or in salt form, into a different compound offormula (I) or an E/Z isomer or tautomer thereof, in each case in freeform or in salt form, separating a mixture of E/Z isomers obtainable inaccordance with the process and isolating the desired isomer, and/orconverting a free compound of formula (I) obtainable in accordance withthe process or by another method, or an E/Z isomer or tautomer thereof,into a salt or converting a salt, obtainable in accordance with theprocess or by another method, of a compound of formula (I) or of an E/Zisomer or tautomer thereof into the free compound of formula (I) or anE/Z isomer or tautomer thereof or into a different salt.
 2. A processfor the preparation of a compound of the formula

in which R₁-R₇ represent, independently of each other, hydrogen or asubstituent; m is 0, 1 or 2; n is 0, 1, 2 or 3; and the bonds markedwith A, B, C, D, E and F indicate, independently of each other, that twoadjacent carbon atoms are connected by a double bond, a single bond, asingle bond and a epoxide bridge of the formula

or a single bond and a methylene bridge of the formula

including, where applicable, an E/Z isomer, a mixture of E/Z isomers,and/or a tautomer thereof, in each case in free form or in salt form,which process comprises 1) bringing a compound of the formula

wherein R₁-R₇, m, n, A, B, C, D, E and F have the same meanings as givenfor formula (III) above, into contact with a biocatalyst that is capableof specifically oxidising the alcohol at position 4″, maintaining saidcontact for a time sufficient for the oxidation reaction to occur andisolating and purifying the compound of formula (III).
 3. A processaccording to claim 1 for the preparation of a compound of the formula(I), in which n is 1; m is 1; A is a double bond; B is single bond or adouble bond, C is a double bond, D is a single bond, E is a double bond,F is a double bond; or a single bond and a epoxy bridge; or a singlebond and a methylene bridge; R₁, R₂ and R₃ are H; R₄ is methyl; R₅ isC₁-C₁₀-alkyl, C₃-C₈-cycloalkyl or C₂-C₁₀-alkenyl; R₆ is H; R₇ is OH; R₈and R₉ are independently of each other H; C₁-C₁₀-alkyl or C₁-C₁₀-acyl;or together form —(CH₂)_(q)—; and q is 4, 5 or
 6. 4. A process accordingto claim 1 for the preparation of a compound of the formula (I), inwhich n is 1; m is 1; A, B, C, E and F are double bonds; D is a singlebond; R₁, R₂, and R₃ are H; R₄ is methyl; R₅ is s-butyl or isopropyl; R₆is H; R₇ is OH; R₈ is methyl R₉ is H.
 5. A process according to claim 1for the preparation of 4″-deoxy-4″-N-methylamino avermectinB_(1a)/B_(1b) benzoate salt.
 6. A process according to claim 1 or 2,wherein the biocatalyst is a microorganism.
 7. A process according toclaim 1 or 2, wherein the biocatalyst is selected from the groupconsisting of a) a living microorganism in the form of vegetative cells,resting cells or freeze dried cells, b) a dead microorganism, preferablyin a partially disintegrated form, with the cell wall/cell membranemechanically or chemically or by spray drying permeabilized, c) crudeextracts of the cell contents of the said microorganism, and d) anenzyme that converts the compounds of the formula (II) into compounds offormula (III), and e) the spores of said microorganism of (a).
 8. Aprocess according to claim 3 or 4, wherein the microorganism is arepresentative of the genus Streptomyces.
 9. A process according toclaim 8, wherein the microorganism is a Streptomyces strain selectedfrom the group consisting of Streptomyces tubercidicus; Streptomyceschattanoogensis, Streptomyces lydicus, Streptomyces saraceticus andStreptomyces kasugaensis.
 10. A process according to claim 9 wherein themicroorganism is the strain Streptomyces R-922 deposited under accessionnumber DSM-13136.
 11. A process according to claim 9 wherein themicroorganism is the strain Streptomyces R-1529 deposited underaccession number DSM-13135.
 12. A process to produce a compound offormula (III) which comprises the following steps: (1) production ofcells by inoculation of a nutrient media promoting cell growth withprecultures of a microorganism capable of converting a compound offormula (II) to a compound of formula (III); (2) harvesting of the cellsafter growth; (3) dissolving a compound of formula (II), in anappropriate solvent; (4) addition of the solution from step (3) to areaction medium which does not promote cell proliferation; (5) additionof cells from step (2) to the reaction medium from step (4); (6) shakingor stirring of the reaction mixture of step (5) in the presence of air;(7) separation of the cells from the medium; (8) extraction of thesupernatant and of the cells with appropriate solvents; (9)concentration of the organic solvent phases from step (8); and (10)purification of a compound of formula (III) contained in the extract (9)by chromatography or crystallisation.
 13. A process to produce acompound of formula (III) which comprises the following steps: (1)dissolving a compound of formula (II) in an appropriate solvent; (2)addition of the solution from step (1) to a nutrient media promotingcell growth; (3) inoculation of the nutrient media of step (2) withprecultures of a microorganism capable of converting a compound offormula (II) to a compound of formula (III); (4) cultivation of amicroorganism capable of converting a compound of formula (II) to acompound of formula (III); (5) separation of the cells from the medium;(6) extraction of the supernatant and of the cells with appropriatesolvents; (7) concentration of the organic solvent phases from step (6)in vacuo; and (8) purification of a compound of formula (III) containedin the extract (7) by chromatography or crystallisation.
 14. A processaccording to claims 12 or 13, wherein the compound of formula (II) isavermectin and the compound of formula (III) is 4″-oxo-avermectin.